The great majority of proteins in mammalian cells are degraded by the ubiquitin (Ub) proteasome pathway (UPS). Although it is generally assumed that rates of protein breakdown by the UPS are determined solely through control of ubiquitination, recent studies have shown that the proteolytic capacity of 26S proteasomes is also tightly regulated and influences rates of proteolysis in cells. We recently made the exciting finding that protein kinase A (PKA) by phosphorylating subunit RPN6 enhances the proteasome?s multiple activities and thereby increases the cell?s capacity to degrade misfolded, aggregation-prone proteins, including mutant proteins that cause Alzheimer?s Disease and ALS. Because of its importance in cell regulation and therapeutic potential, we are pursuing in depth studies of the biochemical mechanisms and physiological importance of 26S phosphorylation by PKA and other protein kinases, as well as studies of the newly discovered ability of several proteasome-binding proteins to stimulate its activities. We hope to understand more fully how PKA enhances proteasome function and influences the structure of its 19S regulatory particle. Proteomic studies are planned to identify the short-lived cell proteins degraded faster upon Rpn6 phosphorylation. A valuable tool in these biochemical and structural studies will be construction by CRISPR gene editing of mutant lines carrying phosphomimetic and phosphodead Rpn6 mutations. Selected studies will test if three other kinases reported to phosphorylate proteasome subunits (e.g. protein Kinase G, CamKinase II, and DYRK2), or others alter proteasome function and protein turnover in similar ways as PKA. cAMP/PKA mediate the actions of many hormones and neurotransmitters, and we recently showed that epinephrine and glucagon trigger proteasome activation in hepatocytes by this mechanism. Upon fasting of mice, in muscle and liver Rpn6 becomes phosphorylated and proteasomes activated, as we also found in human muscles after intense exercise. We plan to explore further these actions, which are of clear physiological interest and demonstrate that surprisingly many major hormones can rapidly enhance protein homeostasis by altering proteasome function. Related studies will probe the mechanisms of proteasome activation by certain 26S-binding proteins. 1) The ZFAND protein, ZNF216, which is induced and essential for muscle atrophy, stimulates the proteasome?s degradative activity. 2) The related ZFAND protein, AIRAP, which is induced in heat shock, may cause the marked activation of 26S proteasomes that we recently discovered occurs rapidly on heat shock. 3) We also recently found that the UBL domain, through which many proteins bind to the 26S, by itself can stimulate proteasome activity. We believe this activation is an important new aspect of the functioning of the DUB Usp14 and of the UBL-UBA shuttling factors (e.g. Rad23) that deliver Ub conjugates to the proteasome.